EP0948630B1 - Cloning and characterization of napsin, an aspartic protease - Google Patents

Cloning and characterization of napsin, an aspartic protease Download PDF

Info

Publication number
EP0948630B1
EP0948630B1 EP97948546A EP97948546A EP0948630B1 EP 0948630 B1 EP0948630 B1 EP 0948630B1 EP 97948546 A EP97948546 A EP 97948546A EP 97948546 A EP97948546 A EP 97948546A EP 0948630 B1 EP0948630 B1 EP 0948630B1
Authority
EP
European Patent Office
Prior art keywords
napsin
antibody
human
sequence
enzyme
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Lifetime
Application number
EP97948546A
Other languages
German (de)
English (en)
French (fr)
Other versions
EP0948630A2 (en
Inventor
Gerald Koelsch
Xinli Lin
Jordan J. N. Tang
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Oklahoma Medical Research Foundation
Original Assignee
Oklahoma Medical Research Foundation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Oklahoma Medical Research Foundation filed Critical Oklahoma Medical Research Foundation
Publication of EP0948630A2 publication Critical patent/EP0948630A2/en
Application granted granted Critical
Publication of EP0948630B1 publication Critical patent/EP0948630B1/en
Anticipated expiration legal-status Critical
Expired - Lifetime legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6478—Aspartic endopeptidases (3.4.23)

Definitions

  • the present invention relates to a previously unknown aspartic protease present in human liver, isolated by cloning of a gene from a human liver cDNA library.
  • aspartic protease family are characterized by the presence of catalytic aspartic acid residues in their active center.
  • Aspartic proteases There are five aspartic proteases known to be present in human body. Pepsin and gastricsin are secreted into the stomach for food digestion. Gastricsin is also present in the seminal plasma. Cathepsin D and cathepsin E are present intracellularly to carry out protein catabolism. Renin, which is present in the plasma, is the key enzyme regulating the angiotensin system and ultimately the blood pressure.
  • Eukaryotic, including human, aspartic proteases are homologous in protein and gene sequences, but have different amino acid and nucleotide sequences.
  • the cDNA and genes of all five human aspartic proteases have been cloned and sequenced. They are synthesized as a single chain zymogen of about 380 residues, which are either secreted or directed to intracellular vacuoles.
  • an N-terminal pro segment of about 45-residues is cleaved off to produce mature enzymes ( Tang and Wong, J. Cell. Biochem. 33, 53-63 (1987) ).
  • mature proteases are further cut into two chains.
  • the three-dimensional structures of the aspartic proteases are very similar.
  • Each enzyme contains two internally homologous lobes ( Tang et al. , Nature 271, 618-621 (1978) ).
  • the active-site cleft which can accommodate eight substrate residues, and two catalytic aspartic acids, are located between the lobes.
  • proteases have distinct and important physiological roles. In addition to their importance in physiological functions, these enzymes are also associated with pathological states. For example, human pepsin and gastricsin are diagnostic indicators for stomach ulcer and cancer ( Samloff, Gastroenterology 96, 586-595 (1989) ; Miki et al., Jpn. J. Cancer Res. 84, 1086-1090 (1993) ). Cathepsin D is located in the lysosome. Its main function is the catabolism of tissue proteins. Recent evidence from mice without a functional cathepsin D gene, however, indicates that this enzyme plays a role in the development of intestine in newborn animals.
  • Cathepsin D is also associated with human breast cancer metastasis ( Rochefort, Acta Oncologica 31, 125-130 (1992) ).
  • Cathepsin E is located in the endoplasmic reticulum of some cells, such as erythrocyte and stomach mucosa cells. It has been applied in the processing of antigens in the immune cells.
  • Human aspartic proteases have important medical uses.
  • the levels of the proenzymes of human pepsinogen and progastricisin present in the bloodstream and the ratio between the two levels is used in the diagnostic screening of human stomach cancer ( Defize, et al., Cancer 59, 952-958 (1987) ; Miki, et al., Jpn. J. Cancer Res. 84, 1086-1090 (1993) ) and ulcer ( Miki, et al., Adv. Exp. Med. Biol. 362, 139-143 (1995) ).
  • the secretion of procathepsin D is elevated in breast cancer tissue.
  • procathepsin D in breast cancer is used for clinical prognosis ( Rochefort, Acta Oncologica 31, 125-130 (1992) ).
  • the analysis of renin in the diagnosis of hypertension is a routine clinical procedure ( Brown et al., Handbook of Hypertension 1, 278-323 Robertson, editor (Elsevier Science Publishers, Amsterdam, 1983 ).
  • napsin A previously unknown aspartic protease capable of cleavage of proteins by hydrolysis, referred to herein as "napsin", has been cloned from a human liver library. Two cDNA clones have been cloned, sequenced and expressed. These encode isozymes of the protease, referred to as “napsin A” and “napsin B". One clone is unusual in that it does not include a stop codon but can be used to express protein. The gene has also be obtained and partially sequenced. A process for rapid purification of the enzyme using immobilized pepstatin has also been developed, and enzyme isolated from human kidney tissue. Polyclonal antibodies to the enzymes have been made which are also useful for isolation and detection of the enzyme.
  • Human napsin A cDNA sequence lacks a stop codon from all clones obtained, yet all features otherwise indicate a functional aspartic protease, including intact active site elements, a conserved Tyr75 (pepsin numbering), and a pro-peptide of approximately 40 amino acids.
  • the characteristic aspartic protease, napsin A contains a C-terminal extension, abundance of proline residues, and an RGD motif (integrin-binding motif) near the surface of the 3-D structure of napsin as judged by homologous crystal structures of mammalian aspartic proteases (i.e., pepsin and cathepsin D).
  • the cDNA of napsin A including the leader peptide and the 3' untranslated region and a stretch of polyadenine, was PCR amplified with primers PLHNAP-FWD (5'- AAGCTTATGTCTCCACCACCGCTGCTGCTACCCTTGCTGC) and PLHNAP-REV (5'- AAGCTTTTATTTTTTTTTTTTTTTTTTCAATGGAAATATTGG) and cloned into the HindIII site of vector pLNCX for expression from the CMV promoter (Dusty Miller). Isolated plasmid was transformed into human kidney 293 cells (ATCC).
  • Cells were recovered (8 - 120 mg) and lysed with 50 mM NaOAc, 20 mM zwittergent, pH 3.5 (NAZ buffer) with vortexing. Lysate was incubated on ice for 1 hour. The supernatant from centrifugation at 14,000 xg was employed directly for detection of expressed Napsin A by addition of a 40 ⁇ l aliquot of pepstatin-A-agarose (Sigma). The sample was rotated in a 50 ml conical tube at 4°C for 1 week. The matrix was settled and washed twice with 20 ml of NAZ buffer, and three times with 20 mM Tris HCl, 0.5 M KCl, pH 8.2 (TK buffer).
  • TK buffer 20 mM Tris HCl, 0.5 M KCl, pH 8.2
  • Genomic clones of human napsin were obtained by screening of a human genomic DNA library, cloned into bacterial artificial chromosomes (pBELO-BAC11) ( Kim et al., Nucl. Acids Res. 20, 1083-1085 (1992) ).
  • the source of genomic DNA for the library was from 978SK and human sperm cell lines, and contained over 140,000 clones.
  • Synthetic oligonucleotide probes were labelled with 32 P :
  • the human napsin A gene is encoded in 9 exons ( Figure 3F ).
  • the exon/intron junctions are clearly defined by both the cDNA sequence and the junction motifs.
  • the human napsin A coding region contains an open reading frame starting from the initiation codon ATG (nucleotide 1 in Figures 1A-1D ) for about 1.2 kb to a polyA stretch in the cDNA sequences.
  • the genomic exon sequence of napsin A do not contain an in-frame stop codon in the entire coding region before the polyA stretch.
  • the absence of a stop codon in napsin A is confirmed.
  • the absence of stop codon has not been observed for the gene of other mammalian proteins.
  • the cDNA (thus the mRNA) of napsin A is present in different human tissues. It was of interest to see if napsin A gene is capable of expressing protein product. These results are described below.
  • Clones 559204 and 163167 expressing human napsin B were obtained from ATCC and partially sequenced as described above.
  • Figures 4A-4E display the resulting full-length DNA sequence encoding Napsin B and the predicted amino acid sequence.
  • Nucleotides 1 - 1191 were obtained from genomic clones (described above for Napsin A) and from 1192 - 1910 from ATCC cDNA clones.
  • the napsin B gene sequence is 92%a identical to that of napsin A, and the putative protein sequence from each exhibits 91 % identity.
  • napsin B protein sequence Similar to napsin A, the deduced napsin B protein sequence possesses typical aspartic protease motifs, and the same c-terminal extension, RGD motif, and proline-rich regions as in the cDNA of napsin A ( Figs. 4A-4E ). Unlike the napsin A gene, napsin B gene has an in-frame stop codon.
  • napsin is related to human cathepsin D, and is similar to mouse aspartic protease-like protein, but the differences are readily apparent.
  • Figure 2D is a diagram of degree of relatedness and also presents the percentage of identical residues.
  • napsin differs as much from other aspartic proteases as they differ from one another.
  • napsin is an aspartic protease.
  • the critical active site aspartic residues at positions 32 and 215 are present in the conversed DTG sequences.
  • (b) The presence of Tyr-75 (Y) and some conserved residues around it indicate a functional 'flap' which is characteristic of aspartic proteases.
  • (c) The pro region corresponding to residues 1p to 44p is present in napsin, indicating that it is a proenzyme of the aspartic protease and is capable of activation.
  • RGD sequence is found at position 315 to 317 (porcine pepsin residue numbers by convention). This motif has been shown to be important in integrin bonding which is related to the regulation of cellular functions such as cell cycle, hemostasis, inflammation and cell proliferation. This sequence may have particular functional meaning for napsin.
  • a napsin-specific polyclonal antiserum was produced using the following procedure.
  • An 18 amino acid epitope of Napsin A which was synthesized as a multiple antignic peptide (MAP) on a poly-lysine backbone by the Molecular Biolgy Resource Facility (OUHSC).
  • This epitope (MKSGARVGLARARPRG) was common to both napsin A and B, and sufficiently dissimilar from cathepsin D, their closest homolog. This region is likely to be located on the surface of Napsin A as determined from the cathepsin D crystal structure coordinates (Erickson, 1993). Aliquots of 1 mg in 1 ml of H 2 O were used to immunize goats (Hybridoma Lab, Oklahoma Medical Research Foundation).
  • Serum collected was ammonium sulfate precipated multiple times (Antibodies Lab manual) and affinity purified using the Napsin A MAP coupled to affi-gel 10 (BioRad). This anti sera was used at 1:5000 dilution in the detection of Napsin A on PVDF membranes transblotted from SDS-PAGE gels (NOVEX). The ECL system (Pierce) was used for detection of primary antibody.
  • Sections of approximately 8 grams of human kidney cortex (Cooperative Human Tissue Network, National Cancer Institute, NIH) were homogenized in a Waring blender in buffer composed of 20 mM Tris HCl, 50 mM NaCl, 20 mM zwittergent, and 1 ⁇ M each of TPCK, TLCK, and EDTA, pH 7.5 (buffer TZ).
  • the homogenate was made 40% ammonium sulfate with gentle stirring, and centrifuged 10,000 xg.
  • the resulting supernatant was made 70 % ammonium sulfate and centrifuged 10,000 xg.
  • the material insoluble in 70% ammonium sulfate (the 40-70% cut) was dissolved in 15 ml of buffer TZ and made pH 4.0 with 30 ml of NAZ buffer. Following incubation on ice for 1 hour, the sample was centrifuged at 14,000 x g. To the resulting supernatant, a 0.1 ml aliquot of pepstatin-A-agarose (Sigma) was added. Detection of napsin B in cell lines followed the procedure outlined above for detection of recombinant napsin A.
  • Napsin B was detected in tissue samples of human kidney cortex and in the human kidney cell line Hut-78: human kidney (0-40% ammonium sulfate cut); human kidney (40-70% cut); Hut-78 cells, in apparently four forms.
  • Hut-78 human kidney (0-40% ammonium sulfate cut
  • N-terminal sequences agreed well with the predicted activation cleavage site in pronapsin B by comparing to the activation cleavage sites in homologous procathepsin D and other aspartic protease zymogens. In the 40-70% ammonium sulfate cut, three forms were detected. A 46-50 kDa single chain form, and two two-chain forms. The 46-50 kDa band produced the same heterogeneous sequence Napsin B sequence as obtained for the larger molecular weight band in the 40% ammonium sulfate cut.
  • the two lower molecular weight fragments of approximately 8 and 4 kDa produced the same amino-terminal sequence (VRLCLSGFQALDVPPPAGPF) corresponding to the C-terimal region of Napsin B.
  • a prominent 40 kDa band of the transblotted preparation was sequenced, and produced the same heterogeneous amino terminal sequence as the 46-50 kDa band, indicating two species of two-chain Napsin B: an 8 kDa and 40 kDa as well as a 4 kDa and a 40 kDa species.
  • a variety of clinical and diagnostic uses for the enzyme can be designed based on analogy to the uses of the related aspartic proteases.
  • the proteins, nucleotide molecules, and methods for isolation and use thereof have a wide variety of applications, particularly in diagnostic applications. Since aspartic proteases are well known to be correlated with certain disorders, such as breast cancer and high blood pressure, and napsin is expressed in the kidney, measurement of the levels and/or types of napsin expressed in tissue, especially kidney, can be correlated with the presence and severity of disorders.
  • the recombinant DNA and reagents derived thereform can be used to assay for napsin expression in healthy and in people inflicted with illness. Napsin sequences can be used to track the presence of napsin genes in patients for possible linkage to diseases.
  • the amount of napsin can be determined using standard screening techniques, ranging from isolation of napsin from the tissue, using for example immobilized anti-napsin (or anti-napsin A or anti-napsin B) or pepstatin, to detection and quantification with labelled antibodies, to determination of the amount of mRNA transcribed in the tissue, using labelled nucleotide probes.
  • Polyclonal antibodies were produced using standard techniques for immunization of an animal with purified protein in combination with an adjuvant such as Freunds' adjuvant. Monoclonal antibodies can also be prepared using standard techniques, for example, by immunizing mice until the antibody titer is sufficiently high, isolating the spleen and doing a fusion, and then screening the hybridomas for those producing the antibodies of interest. These can be antibodies reactive with any napsin, or reactive with napsin A but not B and vice versa.
  • Humanized antibodies for therapeutic applications, and recombinant antibody fragments can also be generated using standard methodology.
  • a humanized antibody is one in which only the antigen-recognition sites or complementarity-determining hypervariable regions (CDRs) are of non-human origin, and all framework regions (FR) of variable domains are products of human genes.
  • CDRs complementarity-determining hypervariable regions
  • FR framework regions
  • RPAS is combined with the CDR grafting method described by Daugherty et al., Nucl. Acids Res., 19:2471-2476 (1991) .
  • variable region DNA of a selected animal recombinant anti-idiotypic ScFv is sequenced by the method of Clackson, T., et al., Nature, 352:624-688 (1991) .
  • animal CDRs are distinguished from animal framework regions (FR) based on locations of the CDRs in known sequences of animal variable genes.
  • FR animal framework regions
  • the CDRs are grafted onto human heavy chain variable region framework by the use of synthetic oligonucleotides and polymerase chain reaction (PCR) recombination. Codons for the animal heavy chain CDRs, as well as the available human heavy chain variable region framework, are built in four (each 100 bases long) oligonucleotides. Using PCR, a grafted DNA sequence of 400 bases is formed that encodes for the recombinant animal CDR/human heavy chain FR protection.
  • PCR polymerase chain reaction
  • CDR-grafted immunoglobulin gene is accomplished by its transfection into human 293 cells (transformed primary embryonic kidney cells, commercially available from American Type Culture Collection, Rockville, MD 20852) which secrete fully grafted antibody. See, e.g., Daugherty, B.L., et al., Nucl. Acids Res., 19:2471-2476, 1991 .
  • humanized ScFv is expressed on the surface of bacteriophage and produced in E. coli as in the RPAS method described below.
  • RPAS Recombinant Phage Antibody System
  • antibody variable heavy and light chain genes are separately amplified from the hybridoma mRNA and cloned into an expression vector.
  • the heavy and light chain domains are co-expressed on the same polypeptide chain after joining with a short linker DNA which codes for a flexible peptide.
  • This assembly generates a single-chain Fv fragment (ScFv) which incorporates the complete antigen-binding domain of the antibody.
  • the ScFv with binding characteristics equivalent to those of the original monoclonal antibody is selected [ See, e.g., McCafferty, J., et al., Nature, 348:552-554 (1990) ; Clackson, T., et al., Nature, 352:624-688 (1991) .
  • the recombinant ScFv includes a considerably smaller number of epitopes than the intact monoclonal antibody, and thereby represents a much weaker immunogenic stimulus when injected into humans.
  • Nucleotide probes can be used to screen for napsin expression or the types and/or ratios of isoforms present. These can be cDNA sequences or other molecules designed based on the sequences reported herein, or which are obtained using standard techniques from libraries generated from different cell types or species. It is understood that while the sequence reported here is of human origin, the same proteases will be present in other species of animals, and will vary to some degree in both the amino acid sequence and the nucleotide sequence. Napsin is referred to herein as an aspartic protease having the naturally occuring amino acid sequence from human or other animals, or a composite sequence constructed by substitution of amino acids from one species into another, at the equivalent position, other than at the active site, discussed above.
  • a nucleotide molecule encoding napsin can be naturally occurring, as described herein, or designed and made synthetically based on the amino acid sequence. Moreover, since at least two isoforms have been identified, it is expected that additional isoforms will be found in tissues other than kidney or liver. These isoforms are intended to encompassed within the term "napsin".
  • Nucleotide molecules can be used to assay for amount, type or a combination thereof, using standard diagnostic techniques.
  • probes will include a segment from a DNA encoding napsin of at least fourteen nucleotides, which should be sufficient to provide specificity under standard hybridization conditions, and even more so under stringent conditions.
  • Reaction conditions for hybridization of an oligonucleotide probe or primer to a nucleic acid sequence vary from oligonucleotide to oligonucleotide, depending on factors such as oligonucleotide length, the number of G and C nucleotides, and the composition of the buffer utilized in the hybridization reaction.
  • Moderately stringent hybridization conditions are generally understood by those skilled in the art as conditions approximately 25°C below the melting temperature of a perfectly base-paired double-stranded DNA. Higher specificity is generally achieved by employing incubation conditions having higher temperatures, in other words more stringent conditions. In general, the longer the sequence or higher the G and C content, the higher the temperature and/or salt concentration required.
  • Chapter 11 of the laboratory manual of Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, second edition, Cold Spring Harbor Laboratory Press, New York (1990 ) describes hybridization conditions for oligonucleotide probes and primers in great detail, including a description of the factors involved and the level of stringency necessary to guarantee hybridization with specificity.
  • the probe should be from 20 to 10,000 nucleotides. Smaller nucleotide sequences (20-100) lend themselves to production by automated organic synthetic techniques. Sequences from 100-10,000 nucleotides can be obtained from appropriate restriction endonuclease treatments. The labeling of the smaller probes with the relatively bulky chemiluminescent moieties may in some cases interfere with the hybridization process.
  • Both antibodies and nucleotide molecules can be labelled with standard techniques, for example, with radiolabels, fluorescent labels, chemiluminescent labels, dyes, enzymes, and other means for detection, such as magnetic particles.
  • selective labeling of the active site with fluorescein can be performed by the method of Bock ( Bock, P.E. (1988) Biochemistry 27, 6633-6639 ).
  • a blocking agent is reacted with enzyme for 1 hour at room temperature.
  • the covalently modified enzyme is incubated at room temperature for one hour with 200 ⁇ M 5-(iodoacetamido)fluorescein (Molecular Probes). Free fluorescein is removed by gel filtration on a PD-10 column (Pharmacia).
  • each molecule of fluoresceinated enzyme contains a single dye at the active site and hence all of the fluorescent molecules behave identically.
  • iodogen Pieris
  • Free 125 I can be removed by gel filtration on a PD-10 column.
  • Recombinant proteins, and fragments thereof, are useful as controls in diagnostic methods.
  • the cDNA and gene sequences of napsin A were determined.
  • the DNA was expressed in a recombinant system (human cell line) and the activity of the enzyme characterized.
  • the cDNA and gene sequences of napsin B were determined.
  • the proteins can be used as standards, or as discussed below, therapeutically as aspartic proteases and in studies of enzyme behavior.
  • the expression of recombinant proteins from a cDNA without stop codon may offer certain advantages.
  • Antibodies and nucleotide probes are primarily useful in the detection of napsin, or its isoforms. In some cases it may also be useful to isolate the purified protein. As described above, a procedure was devised to bind napsin A and napsin B on to a pepstatin-affinity column. Immobilized pepstatin can be used to purify either naturally occurring, or recombinant, napsin, from tissues in which it is expressed, for diagnostic applications.
  • the aspartic proteases may be useful in applications similar to those for which cathepsin D are used. Clinically, it may be advantageous to transfect, even transiently, the gene encoding napsin to treat disorders in which the individual is deficient in the protease, or to transfect an antisense, targeted ribozyme or ribozyme guide sequence, or triple helix to prevent or decrease enzyme expression, in individuals with disorders characterized by elevated levels of enzyme.

Landscapes

  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Biomedical Technology (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
EP97948546A 1996-11-20 1997-11-20 Cloning and characterization of napsin, an aspartic protease Expired - Lifetime EP0948630B1 (en)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
US3119696P 1996-11-20 1996-11-20
US31196P 1996-11-20
US4612697P 1997-05-09 1997-05-09
US46126P 1997-05-09
PCT/US1997/021684 WO1998022597A2 (en) 1996-11-20 1997-11-20 Cloning and characterization of napsin, an aspartic protease

Publications (2)

Publication Number Publication Date
EP0948630A2 EP0948630A2 (en) 1999-10-13
EP0948630B1 true EP0948630B1 (en) 2008-04-23

Family

ID=26706937

Family Applications (1)

Application Number Title Priority Date Filing Date
EP97948546A Expired - Lifetime EP0948630B1 (en) 1996-11-20 1997-11-20 Cloning and characterization of napsin, an aspartic protease

Country Status (11)

Country Link
US (1) US6225103B1 (pt)
EP (1) EP0948630B1 (pt)
JP (2) JP2001503643A (pt)
AT (1) ATE393227T1 (pt)
AU (1) AU734220B2 (pt)
CA (1) CA2271294A1 (pt)
DE (1) DE69738653T2 (pt)
DK (1) DK0948630T3 (pt)
ES (1) ES2308791T3 (pt)
PT (1) PT948630E (pt)
WO (1) WO1998022597A2 (pt)

Families Citing this family (40)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0897986A3 (en) * 1997-08-21 2000-07-05 Smithkline Beecham Corporation Aspartic proteinase 5 (ASP5)
US7045333B1 (en) 1998-01-16 2006-05-16 Incyte Corporation Human protease molecules
US6432690B1 (en) * 1998-01-16 2002-08-13 Incyte Genomics, Inc. Human aspartic proteases
US6203979B1 (en) 1998-01-16 2001-03-20 Incyte Pharmaceuticals, Inc. Human protease molecules
AU5213199A (en) * 1998-07-16 2000-02-07 Incyte Pharmaceuticals, Inc. Human aspartic proteases
EP1939297A1 (en) 1998-09-24 2008-07-02 Pharmacia & Upjohn Company LLC Alzheimer's disease secretase
US7115410B1 (en) 1999-02-10 2006-10-03 Elan Pharmaceuticals, Inc. β-secretase enzyme compositions and methods
US7456007B1 (en) 1998-12-31 2008-11-25 Elan Pharmaceuticals, Inc. β-secretase enzyme compositions and methods
JP4653318B2 (ja) 1999-02-10 2011-03-16 エラン ファーマシューティカルズ，インコーポレイテッド βセクレターゼ酵素組成物および方法
US7514408B1 (en) 1999-12-02 2009-04-07 Elan Pharmaceuticals, Inc. β-secretase enzyme compositions and methods
JP2001172200A (ja) * 1999-12-17 2001-06-26 Meneki Seibutsu Kenkyusho:Kk ヒトナプシンａに対する抗体およびこれを用いる医薬
AU2001267548A1 (en) * 2000-06-26 2002-01-08 Bayer Aktiengesellschaft Regulation of human napsin-like aspartyl protease
EP1666452A2 (en) 2000-06-30 2006-06-07 Elan Pharmaceuticals, Inc. Compounds to treat Alzheimer's disease
PE20020276A1 (es) 2000-06-30 2002-04-06 Elan Pharm Inc COMPUESTOS DE AMINA SUSTITUIDA COMO INHIBIDORES DE ß-SECRETASA PARA EL TRATAMIENTO DE ALZHEIMER
EP1500663A1 (en) * 2000-09-28 2005-01-26 Eli Lilly And Company Secreted proteins and their uses
US7217556B1 (en) 2000-12-23 2007-05-15 Pfizer Inc Crystallization and structure determination of glycosylated human beta secretase, an enzyme implicated in Alzheimer's disease
US7806980B2 (en) * 2000-12-23 2010-10-05 Elan Pharmaceuticals, Inc. Method for crystallizing human beta secretase in complex with an inhibitor
US7601528B1 (en) 2000-12-23 2009-10-13 Elan Pharmaceuticals, Inc. Crystallization and structure determination of glycosylated human beta secretase, an enzyme implicated in alzheimer's disease
US7384773B1 (en) * 2001-05-10 2008-06-10 Pfizer Inc Crystal of HIV protease-cleaved human beta secretase and method for crystallization thereof
US7524668B1 (en) * 2001-05-10 2009-04-28 Elan Pharmaceuticals, Inc. Crystal of human beta secretase having monoclinic space group symmetry C2 and methods for crystallization thereof
AU2002314914A1 (en) 2001-06-01 2002-12-16 Elan Pharmaceuticals, Inc. Hydroxy alkyl amine derivatives as beta-secretase inhibitors and their use for the treatment of alzheimer's disease and similar diseases
MXPA03011521A (es) 2001-06-13 2004-03-18 Elan Pharm Inc Aminadioles para tratamiento de enfermedad de alzheimer.
MXPA04000337A (es) 2001-07-10 2004-07-23 Upjohn Co Amindioles para tratamiento de enfermedad de alzheimer.
MXPA04000338A (es) 2001-07-10 2004-07-23 Upjohn Co Diamindioles para tratamiento de enfermedad de alzheimer.
US7186539B1 (en) 2001-08-31 2007-03-06 Pharmacia & Upjohn Company Method for refolding enzymes
BR0213139A (pt) 2001-10-04 2004-08-10 Elan Pharm Inc Composto, método de tratamento ou prevenção de doenças e de preparação de um composto, uso de um composto e composição farmacêutica
IL161881A0 (en) 2001-11-08 2005-11-20 Upjohn Co N,N'-substituted-1,3-diamino-2-hydroxypropane derivatives
JP4272070B2 (ja) 2002-02-21 2009-06-03 ファルマシア・アンド・アップジョン・カンパニー・エルエルシー 修飾ｂａｃｅ
AU2003303141A1 (en) 2002-04-30 2004-07-22 Elan Pharmaceuticals, Inc. Hydroxypropyl amides for the treatment of alzheimer's disease
US7442537B1 (en) 2002-05-10 2008-10-28 Elan Pharmaceuticals, Inc. Crystals of unliganded beta secretase and/or beta secretase-like proteins and the use thereof
WO2004009797A2 (en) * 2002-07-23 2004-01-29 Incyte Corporation Protein modification and maintenance molecules
US7351738B2 (en) 2002-11-27 2008-04-01 Elan Pharmaceuticals, Inc. Substituted ureas and carbamates
MXPA05011150A (es) 2003-04-21 2005-12-14 Elan Pharm Inc 2-hidroxi-3-diaminoalcanos de benzamida.
US7482136B2 (en) 2003-05-02 2009-01-27 Elan Pharmaceuticals, Inc. Glycosylation variants of BACE
WO2005044977A2 (en) * 2003-06-25 2005-05-19 Diadexus, Inc. Compositions, splice variants and methods relating to lung specific genes and proteins
JP2007528400A (ja) 2004-03-09 2007-10-11 エラン ファーマシューティカルズ，インコーポレイテッド 置換ヒドロキシエチルアミン系のアスパラギン酸プロテアーゼ阻害薬
WO2005095454A1 (en) * 2004-03-25 2005-10-13 Diadexus, Inc. Lng105 antibody composition and methods of use, and use of lng105 to assess lung cancer risk
EP1773756A2 (en) 2004-07-09 2007-04-18 Elan Pharmaceuticals, Inc. Oxime derivative substituted hydroxyethylamine aspartyl protease inhibitors
WO2007047305A1 (en) 2005-10-12 2007-04-26 Elan Pharmaceuticals, Inc. Methods of treating amyloidosis using cyclopropyl derivative aspartyl protease inhibitors
US8222264B2 (en) 2007-07-06 2012-07-17 Boehringer Ingelheim International Gmbh Substituted amino-quinazolinones, medicaments comprising said compound, their use and their method of manufacture

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
IL107250A (en) * 1993-10-12 2001-08-08 Yeda Res & Dev Dna molecules involved in programmed cell death, proteins encoded thereby and methods and compositions using said dna molecules and proteins
GB9618966D0 (en) * 1996-09-11 1996-10-23 Smithkline Beecham Plc Novel compounds
US5776759A (en) * 1996-09-26 1998-07-07 Incyte Pharmaceuticals, Inc. Two novel human cathepsin proteins

Also Published As

Publication number Publication date
AU5459298A (en) 1998-06-10
WO1998022597A3 (en) 1998-07-09
ES2308791T3 (es) 2008-12-01
AU734220B2 (en) 2001-06-07
WO1998022597A2 (en) 1998-05-28
DK0948630T3 (da) 2008-08-11
CA2271294A1 (en) 1998-05-28
JP2008118996A (ja) 2008-05-29
DE69738653T2 (de) 2009-06-04
EP0948630A2 (en) 1999-10-13
PT948630E (pt) 2008-06-11
JP2001503643A (ja) 2001-03-21
DE69738653D1 (de) 2008-06-05
US6225103B1 (en) 2001-05-01
ATE393227T1 (de) 2008-05-15

Similar Documents

Publication Publication Date Title
EP0948630B1 (en) Cloning and characterization of napsin, an aspartic protease
WO1998022597A9 (en) Cloning and characterization of napsin, an aspartic protease
AU699748B2 (en) Recombinant HK2 polypeptide
JP4450767B2 (ja) サイクリックｇｍｐ結合性、サイクリックｇｍｐ特異的ホスホジエステラーゼの材料および方法
JP4205738B2 (ja) 安定ｈｋ２ポリペプチド変異体
WO1995030758A9 (en) Recombinant hk2 polypeptide
WO1998046795A1 (en) Method for detection of recurrent prostate cancer
CA2217492A1 (en) Amyloid precursor protein protease
JP2002501384A (ja) 前立腺特異的抗原の形態およびその検出方法
EP0974057B1 (en) Methods to detect hk2 polypeptides
JP2001503991A (ja) 転移性前立腺癌の検出方法
JPH09235300A (ja) ヒトｔｉｍｐ−３及び抗ヒトｔｉｍｐ−３モノクローナル抗体並びにその用途
WO1998002748A9 (en) Methods to detect hk2 polypeptides
MXPA96005473A (en) Polipeptido hk2 recombina
US20040259113A1 (en) Method for detection of metastatic prostate cancer
WO1992013080A1 (en) Microsomal endopeptidase
MXPA99004504A (en) Method for detection of metastatic prostate cancer

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 19990618

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE

17Q First examination report despatched

Effective date: 20040716

R17C First examination report despatched (corrected)

Effective date: 20040716

GRAP Despatch of communication of intention to grant a patent

Free format text: ORIGINAL CODE: EPIDOSNIGR1

GRAS Grant fee paid

Free format text: ORIGINAL CODE: EPIDOSNIGR3

GRAA (expected) grant

Free format text: ORIGINAL CODE: 0009210

AK Designated contracting states

Kind code of ref document: B1

Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE

REG Reference to a national code

Ref country code: GB

Ref legal event code: FG4D

REG Reference to a national code

Ref country code: CH

Ref legal event code: EP

REF Corresponds to:

Ref document number: 69738653

Country of ref document: DE

Date of ref document: 20080605

Kind code of ref document: P

REG Reference to a national code

Ref country code: PT

Ref legal event code: SC4A

Free format text: AVAILABILITY OF NATIONAL TRANSLATION

Effective date: 20080529

Ref country code: IE

Ref legal event code: FG4D

Free format text: LANGUAGE OF EP DOCUMENT: FRENCH

REG Reference to a national code

Ref country code: GR

Ref legal event code: EP

Ref document number: 20080401671

Country of ref document: GR

REG Reference to a national code

Ref country code: CH

Ref legal event code: NV

Representative=s name: R. A. EGLI & CO. PATENTANWAELTE

REG Reference to a national code

Ref country code: SE

Ref legal event code: TRGR

REG Reference to a national code

Ref country code: DK

Ref legal event code: T3

REG Reference to a national code

Ref country code: ES

Ref legal event code: FG2A

Ref document number: 2308791

Country of ref document: ES

Kind code of ref document: T3

ET Fr: translation filed
PLBE No opposition filed within time limit

Free format text: ORIGINAL CODE: 0009261

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: NO OPPOSITION FILED WITHIN TIME LIMIT

26N No opposition filed

Effective date: 20090126

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: NL

Payment date: 20101110

Year of fee payment: 14

Ref country code: MC

Payment date: 20101026

Year of fee payment: 14

Ref country code: IE

Payment date: 20101110

Year of fee payment: 14

Ref country code: FR

Payment date: 20101123

Year of fee payment: 14

Ref country code: DK

Payment date: 20101110

Year of fee payment: 14

Ref country code: AT

Payment date: 20101110

Year of fee payment: 14

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: PT

Payment date: 20101111

Year of fee payment: 14

Ref country code: LU

Payment date: 20110110

Year of fee payment: 14

Ref country code: FI

Payment date: 20101110

Year of fee payment: 14

Ref country code: DE

Payment date: 20101117

Year of fee payment: 14

Ref country code: CH

Payment date: 20101112

Year of fee payment: 14

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: BE

Payment date: 20101117

Year of fee payment: 14

Ref country code: GR

Payment date: 20101014

Year of fee payment: 14

Ref country code: GB

Payment date: 20101117

Year of fee payment: 14

Ref country code: SE

Payment date: 20101111

Year of fee payment: 14

Ref country code: IT

Payment date: 20101113

Year of fee payment: 14

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: ES

Payment date: 20101217

Year of fee payment: 14

REG Reference to a national code

Ref country code: PT

Ref legal event code: MM4A

Free format text: LAPSE DUE TO NON-PAYMENT OF FEES

Effective date: 20120521

BERE Be: lapsed

Owner name: OKLAHOMA MEDICAL RESEARCH FOUNDATION

Effective date: 20111130

REG Reference to a national code

Ref country code: NL

Ref legal event code: V1

Effective date: 20120601

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: MC

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20111130

REG Reference to a national code

Ref country code: CH

Ref legal event code: PL

REG Reference to a national code

Ref country code: DK

Ref legal event code: EBP

REG Reference to a national code

Ref country code: SE

Ref legal event code: EUG

REG Reference to a national code

Ref country code: GR

Ref legal event code: ML

Ref document number: 20080401671

Country of ref document: GR

Effective date: 20120605

GBPC Gb: european patent ceased through non-payment of renewal fee

Effective date: 20111120

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: NL

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20120601

Ref country code: CH

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20111130

Ref country code: LI

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20111130

REG Reference to a national code

Ref country code: FR

Ref legal event code: ST

Effective date: 20120731

REG Reference to a national code

Ref country code: IE

Ref legal event code: MM4A

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: BE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20111130

Ref country code: FI

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20111120

Ref country code: IT

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20111120

Ref country code: PT

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20120521

Ref country code: GR

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20120605

REG Reference to a national code

Ref country code: DE

Ref legal event code: R119

Ref document number: 69738653

Country of ref document: DE

Effective date: 20120601

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: SE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20111121

Ref country code: GB

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20111120

Ref country code: DK

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20111130

Ref country code: IE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20111120

REG Reference to a national code

Ref country code: AT

Ref legal event code: MM01

Ref document number: 393227

Country of ref document: AT

Kind code of ref document: T

Effective date: 20111120

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: FR

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20111130

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: AT

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20111120

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: LU

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20111120

REG Reference to a national code

Ref country code: ES

Ref legal event code: FD2A

Effective date: 20130603

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: DE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20120601

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: ES

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20111121